SI24318A - New pentadecapeptides stable salts, process for their preparation and their use for the manufacture of pharmaceutical preparations and their use in therapy - Google Patents

Abstract

The present invention describes new stable salts of pentadecapeptides of the formula: Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Ala Gly Leu Val. n BAA, SEQ ID NO: 1, wherein BAA is a basic amino acid and n is 1, 2 or 3. The invention also describes a process for their preparation, a process for their use for the preparation of pharmaceutical formulations in liquid and solid form and for their use for preventing, protection and treatment of diseases and conditions and dietary supplements.

Description

where n is 1,2 or 3, and BAA is a basic amino acid, a process for their preparation, their use for the manufacture of pharmaceutical compositions and their use in therapy.

A technical problem

Numerous pharmacological studies have demonstrated the protective, regenerative and therapeutic action of pentadecapeptide (BPC-157 or bepecin) with the GlyGluProProProGlyLysProAlaAspAspAlaGlyLeuVal amino acid sequence (SEQ ID NO. 1) on human and animal organisms. Until now, this peptide has always been used either in free form or as acetate or as a salt with bases, for example sodium salt. Characteristic of all these forms was a very poor stability in gastric juice, which greatly limits the oral use of these compounds, while reducing their therapeutic value. Therefore, it was essential to produce this pentadecapeptide in a form that is significantly more stable both in gastric juice and at elevated ambient temperature. In such a formulation, the preparation could be used more successfully, especially for oral administration.

For the sake of greater stability, full efficiency would also be expected.

The state of the art

Pentadecapeptide BPC 157 or bepecin (aka: Body Protecting Compound) has the following peptide sequence: GlyGluProProProGlyLysProAlaAspAspAlaGlyLeuVal and is the N-terminal part of the natural BPC protein that is otherwise found in mammalian gastric juice. Natural BPC protein is obtained from human or animal gastric juice by complicated biochemical methods (US Patent 5,288,708). Notwithstanding the demanding work, this method of production is also inappropriate given the uncertain source of raw material and possible contamination with viruses. Therefore, by chemical synthesis (P. Sikiric, R. Ruchman, B. Ruchman, M. Petek, Peptides 1998, Proceed. Of the 25th EPS, Budapest 1998, p. 814; Z. Pflaum and R. Ruchman, Acta Chim. Slov., 2005, 52, 34-39) prepared only Nterminal portion with the above sequence. It was surprising to find that this fragment retains virtually all the effects of the natural BPC protein (EP 0572688 and US Patent 6,268 346). It appears, therefore, that the biological function of the whole protein is largely responsible for the N-terminal moiety with the sequence of 15 amino acids. From a medical point of view, the compound is very interesting because it affects almost all organs at extremely low concentrations (in the range of ng to mg and more / kg body weight) without any toxic or side effects.

Pharmacological studies have shown that pentadecapeptide bepecin (SEQ ID NO. 1) has the following effects on the body:

- ulcers (ulcers) in any part of the gastrointestinal tract (P. Sikiric et al., Life Sci., 1994; 54, PL63-68); (P.Sikiric et al., Exp.Clin.Gastroenterol., 1991,1,17-20).

- anti-inflammatory irrespective of etiology, (P.Sikiric et al., J.Physiol.lParis /, 1997,91,113122);

- against inflammatory disease of the gastrointestinal tract - Chron's disease (Sikiric et al., J. Physiol./Paris/, 2001,95,295-301);

- protective to the liver and pancreas (Prkačin et al., J.Physiol. / Paris /, 2001, 95, 315-324);

- accelerates the healing of burns (D.Mikuš, P.Sikirič et al., Burns 2003, 29, 323-334; Burns, 2001, 127.817-827);

- promotes wound healing (S.Seiwerth, P.Sikirič, J. Physiol./Pariš/, 1997, 91, 173-178;

- radioactive radiation protection (P.Sikirič, M.Petek, R.Ručman, J.Physiol./Paris/ 1993,87,313-327);

- promotes healing of bone fractures (B. Sebečič et al., Bone, 1999, 24,195-202);

- protects mucous membranes under stressful conditions in interaction with adrenergic and dopaminergic systems (P.Sikiric et al., Dig. Dis. Sci., 1997,42,661-671);

- antitumor effect in certain types of tumors (Ascites, melanoma) (P. Sikiric,

M.Petek, R.Ručman, J.Physiol./Paris/,1993, 87. 313-327: S.Radeljak, S.Seivvert,

P. Sikiric, Melanoma Research, 2004, 14 (4), A14-A15);

- antiviral effect on HSV-1 and HSV-2 herpes viruses, viruses, LCM, CMV, influenza A virus and tick-borne encephalitis (P. Sikirič, R. Ručman, M. Petek, J.Physiol./Paris/1993,87,313- 327);

- accelerates the regeneration of broken nerve connections (P. Sikirič et al, Dig.Dis. Sci., 41, 1604-1614; M. Gjurašin et al, Dig. Dis. Sci., 2003, 48, 1879);

- promotes healing of a ruptured Achilles tendon (M. Starešinič, P.Sikirič et al., J. Orthoped. Res., 2003, 21, 976-983)

- eliminates organic disturbances associated with the formation of NO (P.Sikiric et al. Eur.J.Pharm. 1997, 332, 23-33);

The peptide bepecin, as an integral part of the living organism of its own substance, shows no signs of toxicity. Attempts to determine LD ₅₀ in mice and to obtain acute, subchronic, and chronic toxicity data have been unsuccessful because this peptide does not produce any intravenous, oral, or intraperitoneal administration over a wide range of doses of 10 ng to 100 mg / kg body weight. toxic changes.

Similarly, there are no indications of this effect when tested for teratogenicity and genotoxicity (Salmonella microsomal test).

Salts of the synthetic pentadecapeptide BPC-157 are also described in patents EP 0983300 and US 6,288028. These salts are slightly better than their original form, ie free acid or acetate, but they do not yet have satisfactory stability in gastric juice.

The solution to a technical problem

The main problem we need to solve is the stability of the compound at elevated temperature, and above all the stability in gastric juice. The present invention contemplates novel pentadecapeptide salts with basic amino acids having significantly improved thermal stability as well as stability in gastric juice.

Gastric juice is a complex mixture of various compounds, the main ingredients being: water, sodium chloride, hydrochloric acid, mucin, enzymes, especially pepsin and more. Although the gastric juice is predominantly acidic, the pH varies from 1 to 7, exceptionally it may also be higher. Typical pH values are in the range of 2 to 5, which is also the area of maximum pepsin activity that reveals polypeptides, peptides and proteins.

The invention provides novel stable salts of pentadecapeptide of formula (I):

Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val. nBAA

5 10 15

SEQ ID NO 1 where n is 1, 2 or 3, and BAA is a basic amino acid, such as arginine, lysine, ornithine and the like, may be in the L-, D- or DL-form, preferably L-arginine.

The invention also relates to a process for preparing a salt of a pentadecapeptide of formula (I), wherein the pentadecapeptide of the sequence (SEQ ID NO.1) is reacted with the basic amino acid BAA:

Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val + n BAA 15 10 15

SEQ ID NO 1

I

Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val. nBAA 15 10 15

SEQ ID NO 1 in a molar ratio of 1 to 3 moles of basic amino acid per mole of pentadecapeptide in aqueous solution at room temperature, then adjusting the solution to an appropriate pH to form a salt, which is then purified on an HPLC column and converted to a solid form by lyophilization. In the present invention, the pH of the solution obtained from the reaction of 1 mole of basic amino acid with one mole of pentadecapeptide (SEQ ID NO1) is adjusted to 4.1 to 5.0, preferably 4.60 ± 0.05. In the case of reaction of 2 moles of basic amino acid with 1 mol of pentadecapeptide (SEQ ID NO1) to: 6.7 to 7.8, preferably 7.40 ± 0.05. The present invention also relates to a pharmaceutical formulation in solid or liquid form for oral, intravenous, rectal, vaginal, intramuscular, topical or other use containing a therapeutically effective amount of a pentadecapeptide salt of the present invention.

The present invention also relates to a pharmaceutical formulation additionally containing one or more other active substances, preferably antibiotics or antioxidants.

The present invention further relates to a pharmaceutical formulation additionally containing stability enhancing agents from the group of alkali or alkaline earth carbonates or hydrogen carbonates, preferably sodium hydrogen carbonate.

The present invention further relates to a pharmaceutical formulation additionally containing stability enhancing agents selected from the group of sugars such as trehalose, sorbitol or D-mannitol, preferably D-mannitol.

The present invention further relates to a pharmaceutical formulation for use in the prevention, protection and treatment of the following diseases and conditions:

- Stress disorders and abnormalities, ulceration in any part of the gastrointestinal tract, general anti-inflammatory function, gastrointestinal inflammatory disease, Chron's disease and acute pancreatitis;

- organoprotective action: protection against liver and pancreatic lesions, protection of endothelial cells, prevention of adhesion formation, prevention and treatment of myocardial infarction and stroke, protective function in the immune system;

- treatment of viral infections, in particular hepatitis A, several types of herpes, influenza A and infections with ARBO viruses such as ticks encephalitis, West Nile fever, dengue types 1 to 4, cytomegalovirus CMV and LCM virus, feline leukemia virus;

- treatment of melanoma and related tumors;

- accelerating the healing of wounds, burns, bone fractures, regeneration of broken nerve connections, Achilles tendon and muscle, spinal cord injury;

- treatment of organic abnormalities associated with NO formation: hypertension, hypotension, anaphylaxis, circulatory and septic shock, platelet aggregation;

- treatment of neurological diseases and abnormalities: multiple sclerosis, myasthenia gravis, lupus erimatosis, neuropathies, somatosensory nerve dysfunction, asthma, rhinitis, pemphicus and eczema;

- catecholaminergic dysfunction, schizophrenia, dependence on amphetamine, alcohol and drugs;

- prevention and elimination of problems caused by corticosteroids and NSAIDs;

- treatment of wet macular degeneration;

- as original therapy for disorders requiring rapid reorganization of blood circulation;

- in veterinary medicine, to increase the weight gain of animals and to increase the stability of sperm during storage.

The present invention also relates to the use of a pentadecapeptide salt for the manufacture of preparations useful as a dietary supplement.

DETAILED DESCRIPTION OF THE INVENTION

The composition and structure of the new compounds were determined using mass spectra, amino acid analysis, ¹ H-NMR, FTIR and UV spectra. UV spectra were recorded with a Varian Carry 50 spectrometer, FTIR spectra were recorded with a Perkin Elmer 727B spectrometer, mass spectra with an AutoSpec Q spectrometer and ¹ H-NMR spectra with a Bruker Advance DPX500 spectrometer. Specific intensities [a] _D ²⁸ were determined with a Perkin Elmer 141. For the optical recording, the Veho Discovery camera, VMS.001, x30-30 was used in Example 43, and the T-335 infrared camera (FLIR, USA) was used for thermographic recording. FLIR OuickPot program.

The problem described above was solved with new pentadecapeptide salts of bepecin with basic amino acids, which can be in L, D or DL form. Typical representatives of these amino acids are: arginine, lysine, ornithine, 2,4-diaminobutyric acid, 2,6-diaminocaproic acid, 2,6-diaminohexanoic acid, 2,5-diaminopentanoic acid, 2,6-diaminopimelic acid, 2,3- diaminopropionic acid, citrulline, homoarginine, homolysin and the like. The salts may consist of one molecule of pentadecapeptide and one, two or three molecules of basic amino acid. The pH of the aqueous solutions of these salts also depends on this composition, which indirectly affects the stability. Preferably salts with L-arginine are used. They are prepared by reacting the pentadecapeptide SEQ ID NO 1. with basic amino acids in a molar ratio of 1: 1 to 1: 3 (pentadecapeptide: basic amino acid) in aqueous solution by first adding the basic amino acid in excess, then retitri part of the excess with acetic acid solution to the appropriate pH. Optimal pH values for salts with a ratio of 1: 1 from 4.1 to 5.0, preferably 4.60 ± 0.05; in the case of salts with a ratio of 1: 2 from 6.7 to 7.8, preferably 7.40 ± 0.05: no pH value is set for salts with a ratio of 1: 3. The salt solutions were then purified on an HPLC column filled with a reverse carrier. After lyophilization of the corresponding eluate, pentadecapeptide salts of high purity are obtained.

Salts are very easily soluble in water. Salts containing 2 moles of basic amino acids per 1 mol of pentadecapeptide, preferably a salt with L-arginine, i.e. di-L-arginine bepecin (referred to as Arg-BPC), have the most advantageous properties.

The starting compound for the synthesis, that is, pentadecapeptide BPC-157 oz. bepecin is prepared by peptide synthesis on a solid, polymeric carrier as described in the preceding patents EP-0572688, US 6,268346 and in the article Z. Pflaum, R. Ruchman, Acta Chem. Slov., 2005, 52, 34-39. The pentadecapeptide may be in free form, such as ammonium or sodium salt, or may be in the form of acetate or trifluoroacetate.

The stability of the di-L-argininium salt of bepecin (Arg-BPC) at elevated temperature was determined by an accelerated aging method at 50 ° C and relat. humidity of 65%, which presents relatively harsh conditions for a compound with a peptide structure.

Table 1. Declining bepecin content in salts with condition at 50 ° C and rel. humidity 65%:

Substance: 0 days 10 days 30 days 60 days 90 days BPC acetate 99.65% 95.97% 91.98% 85,49% 85.90% BPC Na sol 99.20% 97,22% 96,46% 97.38% 97.14%

Arg-BPC 99,46% 99,29% 99.20% 98.96% Arg-BPC / NaHCOs 99,46% 99.37% 99,29% 99.16% Lys-BPC 99.47 99.24% 99,27% 98.90%

99,07%

99.15%

99.28%

The table also shows that the addition of sodium hydrogen carbonate to the bepecin salt increases its stability at elevated temperature. This supplement will be in the range of 0.5 mg to 3.0 mg per 1 mg bepecin. This property will be taken into account in the composition of the tablets and capsules. A separate experiment with the addition of selected sugars as trehalose or D-mannitol showed a similar increase in stability.

The stability of the bepecin salt with L-arginine in aqueous solutions was determined by incubation of aqueous salt solutions at a concentration of 1 g / 100ml at 50 ° C and by special experiment in very harsh conditions; at 100 ° C, where decomposition was followed for 1 hour.

Table 2. Declining bepecin content in solutions of different salts at 50 ° C over time:

Substance: PH start / About hours 20 hours 65 hours 148 hours 388 hours BPC acetate 3.88 98.89% 71,17% 55,25% 52,55% 21.30% BPC Na sol 8.42 99.01% 98.36% 97.47% N.D. 96,74% Arg-BPC 7.35 99.05% 98.97% 99.04% 99.10% 99.01% Lys-BPC 7.28 99,07% 99,40% 99.09% 99.35% 99.01% Orn-BPC 7.12 100% 99.64% 99.78% 99.59% 99.44%

Table 3. Cooking of peptide salts at 100 ° C for 1 hour:

Substance: start after 1 hour BPC acetate 98.89% 56,80% BPC Na sol 99.01% 98.56% Arg-BPC 99.05% 99.08% (no change!)

The stability of a compound in gastric juice is a very important parameter, especially for peptides that are rapidly degraded in the presence of the pepsin enzyme and in the acidic medium. Higher persistence in gastric juice means a longer period when the compound is available for resorption and its therapeutic action.

Artificial gastric juice illustrates the situation in normal human gastric juice and contains: 0.08 mol of hydrochloric acid, 0.03 mol of sodium chloride and 1.0 g of pepsin in 1000 ml of water.

The investigated peptide salts at a concentration of 10 mg / 5 ml were incubated in artificial gastric juice at pH values of 2.0, 3.0 and 4.0 at 37 ° C.

Substance: Time (hours): 1.0 1.5 2.0 2.5 0 0.5 BPC acetate 100 21.4 8.2 4.75 2.46 2.10 BPC Na sol 100 21.6 6,7 4.4 4.2 2.7 Arg-BPC 100 30,2 13.5 8.2 6.0 4.9 Table 5. Declining bepecin content (rel.%) In jelly. ori juice pH = 3.0: Substance: Time (hours): 0 0.5 1.0 2.0 3.0 4.0 5.0 BPC acetate 100 41,7 26.1 7.8 2.5 1.1 0.08 BPC Na sol 100 81,9 71.6 56,0 40,2 29,7 10.1 Arg-BPC 100 98.1 96.5 93,6 90,0 87,2 84,9 Table 6. Decrease in the content of bepecin irel.%) In jelly. juice at pH = 4.0: Substance: Time (hours): 0 1 2 3 4 5 6 7 8 BPC acetate 100 89,7 81,3 72.5 60.9 56.4 48.8 44.6 38,0 BPC Na sol 100 99,0 98.9 94.6 89,7 78.3 76.3 68.2 60.9 Arg-BPC 100 99,9 99.5 98.7 96.1 89.8 84.6 73.5 67,2

The content of bepecin in the samples tested was determined by the HPLC method in the following system:

column: Reprosil Orpegen C18, 100, 5 μ, 250 x 4.6 mm, mob. phase A: 0.1% trifluoroacetic acid / 5% acetonitrile / water mob. phase B: 0.11% trifluoroacetic acid / 40% acetonitrile / water gradient: from 100% A to 30% A in 25 min. temperature: 25 ° C flow rate: 1.0 ml / min. detection: UV, 210 nm

The results of the stability determinations confirm that the new amino acid salts of bepecin are significantly more stable in all respects than the known salts of this peptide so far, which is a surprisingly great advantage. 0983300 and US 6,288,028, which are otherwise highly prone to polar decomposition product formation (up to 10%, structure not yet established).

Better stability also results in better biological activity as the intact compound is present in the body for a longer time and available for more effective resorption.

Stability in light

Compounds of this type are very sensitive to ultraviolet light. Therefore, an aqueous solution of Arg-BPC (1 g / 100 ml of water) at 20 ° C was irradiated with ultraviolet light of a wavelength of 253.7 nm and measured absorption in the wavelength range of 230 to 350 nm every 10 minutes for 70 minutes. The results presented in Fig. 1. confirm that the compound is stable.

The novel bepecin salts of the present invention can be used as a therapeutic agent, which, together with an inert pharmaceutically acceptable carrier, is converted into a suitable form, such as tablets or capsules, and then used to treat diseases and conditions, organism injuries, wounds , bone and burn fractures, and viral infections as previously identified by pharmacological investigations.

The present invention also provides an oral pharmaceutical formulation comprising the amino acid salt of bepecin, to which various excipients are mixed. As pharmaceutically acceptable excipients, we use a well-known substance in the art of the average skilled in the art. Inert ingredients, respectively. Pharmaceutical excipients are selected from the following groups:

- Fillers such as: anhydrous lactose, microcrystalline cellulose, starch, calcium phosphate, calcium carbonate, maltodextrin, mannitol, trehalose and others;

binders such as: microcrystalline cellulose, hydroxyalkyl celluloses, povidone, cellulose esters, starch or a mixture thereof;

- decomposers such as: starch, croscarmellose sodium crosslink, crospovidone, microcrystalline cellulose, carboxymethyl cellulose sodium and others, usually in the amount of 1-10%.

- stabilizers such as alkali and alkaline earth hydrogen carbonates and certain selected sugars, preferably sodium hydrogen carbonate and D-mannitol, ·· ·· ·· ·· ... ..

- lubricants and sliding enhancing agents for tabletting: talc, magnesium stearate, stearic acid, potassium stearate and colloidal silica. These substances are usually added to the other ingredients in the final phase.

After comparing the stability of bepecin salts in solutions with different acidity, these compounds are found to be most stable in slightly acidic, neutral or even slightly alkaline media, preferably in the pH range from 6.5 to 8.5. It is therefore appropriate to consider this in the composition of pharmaceutical formulations. For the preparation of tablets for tabletting and encapsulation, the addition of slightly alkaline substances such as alkali and alkaline earth hydrogen carbonates and carbonates, basic carbonates and oxides, such as sodium hydrogen carbonate, calcium hydrogen carbonate and magnesium oxide, is further advantageous. The addition of selected sugars serves to counteract the Maillard reaction. Pharmaceutical formulations are prepared by methods known as direct mixing, dry granulation, wet granulation, or by spraying a solution of bepecin salt on inert pharmaceuticals while drying under hot air. The granulate thus obtained is further added, if necessary, by other ingredients, homogenized and tableted or encapsulated in the usual manner. Pills, capsules or dragees can also be coated with a protective film that is resistant to the action of gastric juice by releasing bepecin only in the intestine.

The amino acid component in the present new salts is responsible for the increased hydrophobicity of the active ingredient in this form. Therefore, a realistic transition to biological membranes is expected, which also allows the preparation of a preparation for action through the skin - for example creams, ointments or patches.

The transdermal system consists of the back of the active substance-impermeable polymer layer, which serves as a reservoir for the active substance and is sensitive to pressure and a protective foil that is perforated in several places to penetrate the active substance and the outer protective foils.

The amino acid salts of bepecin can also be used in the form of a solution, an enema, an injection or a dry injection. In this case, it is used as a solvent or aqueous buffer solution with an optimum pH between 6 and 8, preferably between 7.0 and 7.5. The solution may be sterilized by fine filtration through a 0.22 µm pore filter or very briefly heated to 100 ° C. Dry injection is prepared by freezing in sterile solution containing bepecin salt, buffer and preservative in ampoules and lyophilizing.

The eye or nasal drop solution is prepared using an isotonic and isohydric solution with a pH of 7.0 to 7.5, which contains buffer and preservative in addition to the active substance, bepecin salt, and is sterile filtered.

For the preparation of suppositories for rectal and vaginal use, gelatin, cocoa butter, low-melting natural or semi-synthetic fats, paraffin, glycerin, polyethylene glycols with a molar mass of 1000 to 6000 in which the aqueous solution of the amino acid salt of bepecin is mixed are taken as the basis. Emulsifiers, antioxidants and preservatives are also added. Bepecin salts can also be used as a cream, ointment or gel. In this case, we take the lipophilic, hydrophilic or amphiphilic fat base based on natural vegetable or animal fats, natural oils, to which preservatives, colorants, preservatives, emulsifiers, water or buffer solutions and antioxidants are added.

Bepecin is a compound that works in very low doses. When administered orally, therapeutic doses range from 10 ' ⁵ to 10' ² mg / kg body weight, depending on the type and severity of the disease. The usual dose in tablets, dragees or capsules will be from 0.1 to a maximum of 5 mg. When used locally, concentrations are higher, from 0.001% to 0.5%. Determining the optimal dose is a matter of judgment and experience. It is very important that bepecin does not show any side effects or toxicity. In view of this and its many beneficial effects on the body, it is also possible to use it as a dietary supplement in very low doses, from 1 pg to a maximum of 0.1 mg daily. For this purpose, formulations can also be combined with vitamins, minerals and other beneficial substances.

Other active ingredients which enhance the basic function of bepecin or act synergistically may be added to the present invention.

- substances with antibiotic activity, such as gentamicin, azithromycin, ampicillin, cephalosporins and doxocycline, especially in formulations for topical use where antiviral and antibiotic action is desired. One example is tick ticks where our purpose is to prevent both borelia and tick-borne encephalitis;

- Antioxidant substances, such as green tea dry extract, coenzymeQio, idebenone, curcumin, abigenol, picnogenol and others.

Particularly interesting is the combination with coenzyme Qi ₀ or ubiquinone, which is a very effective antioxidant and free radical scavenger, and can also be used in heart disease related to decreased blood flow, high blood pressure and signs · · · · · - · «« heart failure. Curcumin is also a very important antioxidant lately, which also has a strong anticancer effect.

Our research has shown that bepecin and its salts have a very beneficial effect in the treatment of nervous system diseases, such as multiple sclerosis. It is an inflammatory disease that causes damage to the myelin sheath of the nervous system in the brain and spinal cord. Nerve cells communicate by sending electrical signals through nerve fibers - axons, where the myelin sheath serves as an insulator. In the case of damage to the myelin sheaths, the transmission of electrical signals is interrupted or severely disrupted. In multiple sclerosis, the body's own immune system attacks and damages the myelin sheaths.

There is no really effective cure for multiple sclerosis. Existing therapy attempts to improve the condition after the onset of the disease, prevent new attacks, but is usually associated with various side effects or is poorly accepted.

Bepecin has anti-inflammatory, reparative, protective and immunomodulatory activity.

Based on the results of pharmacological investigations, it is legitimate to expect a good therapeutic effect of bepecin for multiple sclerosis. The formulation with the bepecin L-arginine salt formulation in the adjuvant treatment of secondary lesions and disorders resulting from infection with Lyme borreliosis (L. Reik et al., Neurology, 46, 1989, 790 - 795) or syphilis (also possible) could also be useful ( D. Gray, The Lancet, July 12, 1986, 75 77). In all these cases, damage to the myelin sheaths and lesions in the brain occurs, which is evident by MRI.

Oral preparations with salts of bepecin in stabilized form and in doses of 1 mg to 5 mg will be used in the treatment of multiple sclerosis. These preparations do not cause any harmful or unpleasant concomitant effects and are therefore completely safe for long-term use.

Based on the results of animal pharmacological experiments, we consider the use of bepecin in the form of an appropriate pharmaceutical formulation to be of great benefit in other diseases of the neural etiology such as neuropathy, myasthenia gravis and others.

Bepecin also has a very beneficial effect in the reorganization of collateral blood vessels after serious damage to the femoral artery.

The invention is explained by the following embodiments, which are intended to illustrate the process and in no way limit the invention. Examples 18 to 44 describe experiments to determine the pharmacological activity of bepecin. They were made using ·· ·· ·· ·· ···. · Various in practice widely used experimental models “in-vitro” and “in-vivo”, citing literature sources. Bepecin was used as a salt with L-arginine.

Short description of the pictures

FIG. 1. Demonstration of the stability of the bepecin solution under ultraviolet irradiation, the absorbance of the sample from 1 to 7 is measured every 10 min.

Fig 2. Demonstration of the action of bepecin on B-16 melanoma cells.

FIG. 3. Demonstration of the effect of bepecin on tick-borne encephalitis virus (ΤΒΕ):

A: Simultaneous administration of 10 ² TBE virus (icv) and saline 0.9% NaCI (ip), without bepecin.

B: concurrent administration of 10 ² TBE virus (icv) and bepecin 10pg / kg (ip).

Symptoms of infection are much later.

C: Administration of bepecin 10pg / kg (ip) 4 hours before infection with 10 ² TBE virus (icv). There are no clinical signs of the disease. After 30 days, the animals are killed and some of the brain homogenizate transferred to the new animal, see: D.

D: concurrent brain homogenisate application and new 10 ² TBE (icv) infection Even after 50 days, there are no signs of disease.

FIG. 4. ¹ H-NMR spectra of bepecin salts recorded in D ₂ O:

A - spectrum of bepecin L-lysine salt (1: 3),

B - spectrum of bepecin L-argininium salt (1: 2),

C - spectrum of bepecin L-ornithine salt (1: 2).

FIG. 5. Presentation of collateral blood vessels:

A: control group that received only a local saline bath for 1 min where no collateral vascular lesions appeared;

B: test group that received a bepecin bath for 1 min- shows a rich collateral vasculature of the vasculature with full interconnections.

EXAMPLES

EXAMPLE 1 Preparation of Bepecin D-Arginine Salt (1: 2)

Bepecin trifluoroacetate (500 mg, 0.34 mmol) was dissolved in 25 ml of water, D-arginine (212 mg, 1.22 mmol) was gradually added while stirring and stirred for a further 15 minutes. The pH of the solution was then adjusted to 7.40 ± 0.05 with dilute acetic acid (50%). The solution is filtered, frozen, and lyophilized. The lyophilisate was dissolved in a small amount of water and applied to the start of the HPLC column (diameter = 2.2 cm, height = 25 cm, sorbent Reprosil C18, 10 μ), previously washed and equilibrated with clear water. We elute with increasing ·· ·· · · · · ·· <

isopropanol concentration in water: 0 to 5% isopropanol within 30 min. Initial fractions containing salts and impurities are decided. The major fraction was collected and lyophilized. 550 mg of dry salt are obtained containing 2 moles of D-arginine per 1 mol of pentadecapeptide.

Amino acid analysis corresponds to the composition of:

2-Ala, 1-Val, 3-Gly, 4-Pro, 1-Leu, 2-Asp, 1-Glu, 1-Lys, 2-Arg.

MS (TOF MS ES +): = 1419.8 (pentadecapeptide + H)

M ₂ = 175.1 (D-arginine + H)

FTIR in (cm ¹ ): 3275, 3057, 2958, 2872, 1628, 1533, 1448, 1394, 1315, 1244, 1204, 1161, 1096, 1044, 919, 875.

Specific gravity (c = 1 g / 100 ml) [a] _D ²⁸ = -154 °. HPLC purity: 99.48%.

EXAMPLE 2 Preparation of Bepecin L-argininium Salt (1: 1)

Prepare a solution of bepecin acetate and L-arginine in the same manner as described in Example 1. Adjust the pH of the solution to 4.60 ± 0.05 with dilute acetic acid (50%). The solution was then treated as in Example 1. 232 mg of an amorphous substance containing 1 mole of L-arginine per 1 mole of bepecin was obtained.

Amino acid analysis corresponds to the composition of:

2-Ala, 1-Val, 3-Gly, 4-Pro, 1-Leu, 2-Asp, 1-Glu, 1-Lys, 1-Arg.

MS (TOF MS ES +): _Μ Ί = 1419.8 (pentadecapeptides + H)

M ₂ = 175.1 (L-arginine + H)

FTIR in (cm ¹ ): 3265.3060, 2960, 2875, 1629 (CONH), 1534, 1448, 1394, 1312, 1243, 1202, 1160, 1043, 919, 873.

Specific gravity (c = 1 g / 100 ml) [a] o ²⁸ = -119.0 °. HPLC purity: 98.57%.

EXAMPLE 3 Preparation of Bepecin L-argininium Salt (1: 2) (Arg-BPC)

Bepecin acetate (1 g, 0.705 mmol) and L-arginine (420 mg, 2.41 mmol) were dissolved in 4 ml of water (pH value of the solution was 8.92) and stirred for 20 minutes. The pH was then adjusted to 7.40 ± 0.05 with dilute acetic acid (50%). This solution was then applied to the start of the HPLC column (2.2 cm in diameter, 25 cm in height, sorbent: Reprosil C18, 10μ) previously well washed and moistened with clean water. Elute gradient with increasing concentration of isopropanol in water: from 0% to 5% isopropanol within 30 min. Fractions containing salts and impurities are discarded. The major fraction was lyophilized. 1.10 g of a white amorphous salt, readily soluble in water, are obtained.

Amino acid analysis corresponds to the composition of:

2-Ala, 1-Val, 3-Gly, 4-Pro, 1-Leu, 2-Asp, 1-Glu, 1-Lys, 2-Arg.

MS (TOF MS ES +): M! = 1419.8 (pentadecapeptide + H)

M ₂ = 175.1 (L-arginine + H)

FTIR in (cm ' ¹ ): 3271, 3057, 2958, 2875, 1632 (CONH), 1539, 1448, 1394, 1244, 1205, 1161, 1097, 1044, 919, 873,652

Specific gravity (c = 1 g / 100 ml) [a] _D ²⁸ = -124.3 °. HPLC purity: 99.54%.

^{The 1} H-NMR spectrum is shown in Fig. 4 / B.

EXAMPLE 4 Preparation of Bepecin L Lysine Salt (1: 3) (Lys-BPC)

Bepecin acetate (500 mg, 0.34 mmol) and L-lysine (176 mg, 1.2 mmol) were dissolved in 3 ml of water and stirred for 20 minutes. The solution was then applied to an HPLC column and purified as described in Example 3. 283 mg of a white, water-soluble amorphous solid was obtained.

Amino acid analysis corresponds to the composition of:

2-Ala, 1-Val, 3-Gly, 4-Pro, 1-Leu, 2-Asp, 1-Glu, 4-Lys

MS (TOF MS ES +): Mi = 1419.8 (pentadecapeptide + H)

M ₂ = 147.1 (L-lysine + H)

FTIR in (cm ' ¹ ): 3269, 3060, 2958, 2875, 1981, 1627 (CONH), 1532, 1447, 1393, 1315, 1241, 1203, 1160, 1008, 919, 873, 654

Specific gravity (c = 1 g / 100 ml) [a] _D ²⁸ = -130 °. HPLC purity: 99.47%.

^{The 1} H-NMR spectrum is shown in Fig. 4 / A.

EXAMPLE 5 Preparation of Bepecin L ornithinium Salt (1: 2), (orn-BPC)

Bepecin acetate (500 mg, 0.34 mmol) and L-ornithine (177 mg, 1.05 mmol) were dissolved in 5 ml of water and the pH was adjusted to 7.40 ± 0.05 with dilute acetic acid. The solution was then purified on an HPLC column as described above. 412 mg of white amorphous water-soluble amorphous material are obtained.

Amino acid analysis corresponds to the composition of:

2-Ala, 1-Val, 3-Gly, 4-Pro, 1-Leu, 2-Asp, 1-Glu, 1-Lys, 2-Orn

MS (TOF MS ES +): M! = 1419.8 (pentadecapeptide + H)

M ₂ = 170.1 (L-Orn + H)

FTIR in (cm ' ¹ ): 3269, 2958, 2875, 2162, 1636 (CONH), 1522, 1442, 1389, 1311,

1243, 1203, 1160, 1094, 1040, 911, 873, 654, 610

Specific gravity (c = 1 g / 100 ml) [a] _D ²⁸ = -137 °. HPLC purity: 99.30%.

^{The 1} H-NMR spectrum is shown in Fig. 4 / C.

EXAMPLE 6. Tablets

Ingredient: mg / tablet:

Arg-BPC 1.00

Avicel Cellulose 20.04

Lactose anhydrous 60.53

Sodium hydrogen carbonate 2.25

Crospovidone 5.25

Colloidal silica 0.33

Magnesium stearate_0,60

90.00 mg

EXAMPLE 7. Tablets - film coated

Ingredient: mg / tablet:

Arg-BPC 2.00

Trehalose 96.20

Hydroxypropylcellulose 11.20

Sodium hydrogen carbonate 2.60

Curcumin 5.00

Croscarmellose At 2.00

Magnesium stearate_1,20

Coating resistant to jelly. juice:

EudragitSIOO 0.48

Ammonium hydroxide, 1 mol / l 0.25

Triethylcitrate 0.24

Talk 0.16

Water 2.90

EXAMPLE 8. Eye drops Ingredients:

Arg-BPC 0.50 mg

Sodium phosphate buffer 0.05 mol / l, pH 7.4 85 ml

Sodium chloride 0,44 g

Benzyl alcohol_0,20 ml

Water to total

100 ml

EXAMPLE 9. Capsules Ingridients: mg / capsule: Lys-BPC 1.0 Sodium hydrogen carbonate 2.5 Lactose monohydrate 80.6 Corn starch pregelatinized 15.4 Maanezi's stearate 0.5 100 mg EXAMPLE 10. A solution for topical use Ingridients: g / 100 ml: Water for injections 20,00 Arg-BPC 0.05 Glycerol 22,00 Benzalkonium chloride 0.02 Sodium phosphate buffer, C, 05 mol / l, pH 6, up to 100 ml. EXAMPLE 11. Gel Ingridients: g / 100 g Water inj. 70,00 Arg-BPC 0.20 Carbopol 974, Noveon ^R 1.00 Methyl-p-hydroxybenzoate 0.18 Propyl p-hydroxybenzoate 0.02 Sodium hydroxide 0.01 mol / l to adjust pH to 7.0 Water injection to: 100 ml total EXAMPLE 12. Cream Lipophilic ingredients for 100 g of cream: stearic acid, cetearyl and stearyl alcohol, saturated medium chain caprylic triglycerides and capric acid, silicone oil Dimethicone and sodium cetearyl sulfate, total 16.4 g, melt at 60 ° C. Hydrophilic Ingredients: Propylene glycol (3 g) is added to sterile filtered pure water (70 g) and preservative butyl 4-hydroxybenzoate (0.5 g), mixed and heated to 60 ° C. Z 0.1

The molar sodium hydroxide solution was adjusted to pH 6.5.

The hydrophilic phase is then added to the lipophilic phase while stirring, cooled and stirred until 30-35 ° C is reached. To this mixture was added the antioxidant tocopherol acetate (0.05

g), mix well, and finally add 10 g of an aqueous solution containing 0.1 g of Arg-BPC. Mix well and fill in suitable containers.

EXAMPLE 13. Suppositories

Ingredient: mg / sup. Arg-BPC 0.5 Water 25,0 Glycerol 30.5 Cocoa butter 180,0 Methyl-p-hydroxybenzoate 2.0 Polyethylene glycol PEG 1000 61.0 Tocopherol 1.0 300 mg EXAMPLE 14 Orodispersive tablets Ingredient: mg / tab Arg-BPC 0.5 Starlac ^R 48,9 Acesulfam K 0.4 Magnesium stearate 0.1 Colloidal silica OJ

50,0 mg

EXAMPLE 15. Transdermal preparation - patch

Active Ingredients: Arg-BPC (5.0 mg) and doxocillin (1.0 g), dissolved in a mixture of water (10 g), diethylene glycol monoethyl ether (1.8 g), polyethylene glycol monolaurate (0.5 g) and diethanolamine ( 0.2 g), Add 25 g of polymeric GEL VA solution. RTM.2484 (Monsanto) and mix well. The solution is then allowed to stand for 20 min to vent. It is then applied to an impermeable membrane (polyethylene film or aluminised polyethylene film, for example: 3M-Scotchpack 1006) and dried at 40 to 50 ° C. This layer is then covered with a porous controlled release film and a protective film and cut to a suitable size.

EXAMPLE 16. Extended stability tablets

Restatement:

Bepecin L-argininium salt (1: 2) (1.0 g) and D-mannitol solution in 20 ml of water, sterile • · ·· - · «· ..

filtered (0.22 μ filter) and lyophilized. A white amorphous powder containing 50% of bepecin salt is obtained.

Tablet preparation:

Ingredient: mg / tablet Reformulated Arg-BPC (50%) 2.0 D-mannitol pharm. (Pearlitol 300DC) 70,0 Avicel Cellulose (PH 102) 25,0 Sodium hydrogen carbonate 1.3 Colloidal silica (Aerosil200) 0.7 Maanezi's stearate LP

100 mg

Stability test:

The tablets prepared according to the above procedure were incubated at -15 ° C, 25 ° C and at 50 ° C / 65% relat. humidity. Bepecin content was determined by HPLC method. Incubation time: 18 months.

Temperature: -15 ° C 25 ° C 50 ° C

Bepecin content: 99.8% 96.6% 92.35%

Using the extrapolation method, we can conclude that the tablets will be stable for at least 2 years at room temperature.

EXAMPLE 17. Nutritional supplement with bepecin - effervescent tablets

Ingredient: mg / tablet: Arg-BPC 0.1 Glucose 150,0 Fructose 29.0 Vitamin C 50.4 Sodium hydrogen carbonate 30.0 Natural aroma 0.5 260 mg

EXAMPLE 18. Operation on the NO system

Nitric oxide (NO) plays the role of a signaling molecule in endothelial and nerve cells, as well as as a so-called killer molecule activated by immune cells. In general, both in excess and in NO deficiency, NO appears to contribute greatly to the occurrence of various abnormalities and disorders in the body, such as: hypertension, angina, impotence, circulatory and septic shock, cardiac defects, arrhythmias, stroke, inflammatory processes, adhesion and aggregation of platelets and leukocytes, poor healing of wounds and burns, damage to muscles, tendons, ligaments and bone fractures, gastrointestinal lesions, diabetes, pancreatitis, circulatory and septic shock, arrhythmias, endothelial disorders, heart defects and Parkinson's disease.

Bepecin has the characteristic property of affecting the release and normalization of NO levels and acts against NO blocking agents such as L-NAME and against the increased concentration of NO precursors such as L-arginine. Likewise, the action of bepecin on the NO system also explains to some extent the very wide range of its pharmacological efficacy.

By the methods described in the literature: Regul. Pept. , 2009, 156 (1-3) .83-89: J. Pharm. Sci., 2008, 108 (1), 7-17, and Eur. J. Pharmacol., 1997, 332 (1), 23-33, experimentally demonstrated that bepecin can induce NO "in-vitro" release and counteract the effects of L-NAME. Thus, it normalizes blood pressure disorders that are subsequently caused by NO. Bepecin also protects against arrhythmias caused by methyldigoxin (6 mg / kg, i.v. or i.p.) when used at doses of 10 ng to 10pg / kg body weight, parenterally or orally. It prevents the deterioration of lesions caused by the NO-blocker L-NAME. Bepecin acts on both high and low NO levels and normalizes it.

Bepecin usually acts modulatory in the NO system when used at doses of 10 ng to 10 pg / kg tel. heavier. Prevents worsening of lesions caused by NOS blocker LNAME.

EXAMPLE 19. Effect on wound healing

Using the methods detailed in the literature: J. Physiol. Pharmacol., 2009, 60 (Sup. 7), 191-196; J. Orthop. Res., 2010, 28 (9), 1155-1161; Burns 2005, 31 (3), 310315; Bone, 1999, 24 (3), 195-202; Surg. Today, 2007; 37 (9), 768-777. We found that bepecin, by its action on new tissue formation, angiogenesis and collagen production, significantly accelerates healing:

- cuts in the skin,

- deep skin burns,

- various anastomoses as intestinal wounds,

- different fistulas,

- various cut tissues, in particular ligaments, tendons, muscles and nerves,

- bone fractures, including concurrent soft tissue damage,

- corneal wounds, including abrasion of the entire epithelium or corneal ulceration.

Based on these results and the great similarity of healing processes in rats and humans, bepecin will be able to be successfully used for healing wounds, burns and bone fractures in humans.

EXAMPLE 20. Endothelial protection, angiogenesis, thrombosis, and bleeding disorders

Based on long-known publications (J. Physiol. Paris, 1993, 87, 313-27; Inflammopharmacol., 2006, 14, 214-21; Curr. Pharm. Des., 2010, 16, 1224-34, Bepecin regulates and maintains the endothelial state after intragastric intake of absolute ethanol (ie, cytoprotective activity), and beepecin affects the healing of injuries both inside and outside the gastrointestinal tissue. that after an abdominal aortic anastomosis in rats, the use of bepecin prevents thrombus blockage from occurring for at least 24 hours, but when administered in the case of pre-existing thrombosis and vascular blockage, this thrombus blockage rapidly disintegrates, while maintaining the function of the lower limb.

In other experiments in rats after amputation, we found that bepecin consistently works against prolonged bleeding and thrombocytopenia. Even if long haemorrhage and thrombocytopenia result from aspirin, warfarin and heparin. Bepecin was given intraperitoneally, intravenously or intragastrically (10 ng / kg, 10 pg / kg body weight). Important finding in preventing and eliminating pre-existing clot, prolonged bleeding or thrombocytopenia resulting from the use of really high doses of heparin (250 mg / kg, 25 mg / kg and 10 mg / kg, iv), warfarin (1.5 mg / kg, ia, once daily for 3 consecutive days) and aspirin (0.1 g / kg, ig, once daily for 3 consecutive days, or 1 g / kg, ip, once) was that bepecin alone does not act on coagulation parameters. In view of all this, bepecin therapy could be extended to all important conditions where thrombosis and circulatory abnormalities occur. Bepecin could also be an original therapy for conditions where the egr-1 gene is disrupted.

EXAMPLE 21. Formation of adhesions

Our previous studies have shown a marked effect of bepecin on inflammatory processes and their consequences. In addition, the therapeutic effect of bepecin on adhesion (adhesion) formation and adhesion protection (or at least a significant reduction) was found.

By surgical removal of the parietal peritoneum in rats according to the methods described in Gastroenterology, 2010, 138 (5, Suppl.1), 753; ileo-ileal anastomoses according to Surg. Today, 2007; 37 (9), 768-777; jejuno-ileal anastomoses according to Dig. Dis. Sci., 2009, 54 (10), 2070-2083; we found that bepecin generally reduced adhesion formation (macroscopically and microscopically) combined with functional improvements (anastomosis enhancement, weight gain of animals to normal levels). In this case, bepecin was used in the form of salts with L-arginine (2; 1) and at low doses of 10 pg to 10 ng / kg body weight, parenterally, per axis or topically.

The similarity between the animal model and the conditions in the human body in which adhesion antagonism occurs allows the therapeutic use of bepecin for this purpose.

EXAMPLE 22. Immunomodulation

Immunomodulation (especially as a vital function of macrophages) caused by natural or synthetic substances is considered as a good alternative for the prevention and treatment of both infectious and neoplastic diseases.

Using the method described in J. Physiol. Pharmacol., 2009, 60, Suppl.2, 69, determined the in-vivo efficacy of bepecin in enhancing macrophage activity as major immunologically active cells in mice (ie, increased macrophage motility, restoration of M / P ratio between mononuclear and polynuclear leukocytes) . Mice were injected daily with 50 oz. 100 pg / kg 1.1. (i.p.) bepecin for three consecutive days. Activated macrophages trigger the production and release of factors that regulate B-, T-, and NK-cell function, which is important for immunomodulatory activity. We may use this immunomodulatory effect of bepecin to assist in the treatment of certain infectious or neoplastic diseases.

EXAMPLE 23. NSAIDs - NSAIDs

As previously noted, bepecin has a potent effect on all inflammatory parameters, while greatly improving healing and preventing lesions and injuries.

Paracetamol, aspirin, diclofenac, ibuprofen and similar compounds are toxic as well as at regular doses and are toxic and have numerous side effects reported in the literature: J. Physiol. Pharmacol., 2010, 61 (2), 241-250; Gastroenterology, 2010, 138 (5, Suppl.1),

S-369. This is reflected in particular in the formation of gastric, intestinal and liver lesions, hemorrhages, increases in bilirubin, AST and ALT enzymes, liver encephalopathy and more. Arg-BPC effectively antagonizes increased bleeding after administration of aspirin (1 g / kg ip) or diclofenac (12.5 mg / kg tt, ip) at very low doses of 10 pg to 10 ng / kg tt given orally, intragastric or right in the drinking water.

Arg-BPC works against the side effects of NSAIDs. Moreover, given its good effects on acute and chronic inflammation (where NSAIDs are also used), it would be best to use it alone without NSAIDs.

EXAMPLE 24. Anaphylaxis and anaphylactoid reaction

Serious anaphylactoid reactions occur in the rat in parenteral administration of egg white and dextran in rats (BN Halper, Histamine, Ciba Found. Symp., J. and A. Churcil Ltd., London, 1956, 92-123 and H. Selye, Endocrinology 1937,21,169). Natural or synthetic polymers cause massive release of endogenous histamine. Anesthetized animals received intravenous dextran solutions in concentrations of 6%, 10%, 20%, 40%, 60%, 80% and 90%, and / or egg white (1 ml / rat or 0.15 ml per mouse) in rep. There was marked edema, comprising the cheek, upper and lower lip, snout, paws and genitals (expressed by extreme cyanosis) and poor respiration, and several deaths following dextran and / or egg white administration.

In contrast, bepecin in the dosage range (10 pg, 1 pg, 10 ng, 10 pg / kg) prevents anaphylactoid reaction and vascular collapse. It also significantly improves the action of antihistamines such as clomipramine (20 mg / kg) and cimetidine (10 mg / kg), which are otherwise poorly or not effective under these conditions.

Considering that the models used are regular and mimic the anaphylactoid reaction in humans, the favorable action of bepecin under anaphylactoid conditions is guaranteed.

EXAMPLE 25. Corticosteroids

Corticosteroids, especially when administered systemically, can cause serious disorders (immunosuppression, poor wound healing, etc.) using the method described in the literature (Med. Sci. Monit., 2010, 16 (3), BR 81-88; and Burns. 2005, 31 (3), 310-315) found that bepecin completely prevented poor wound healing resulting from the use of corticosteroids (i.e. 6-alpha-methylprednisolone, 1 mg and 10 mg / kg body weight, ip in mice and rats), activity has been consistently demonstrated in the case of corticosteroids with systematically treated muscle congestion, deep skin burns, severed Achilles tendons and middle collateral ligaments, various fistulas, gastric, intestinal and skin lesions, or corticosteroid immunosuppression (investigation of spleen cells).

Bepecin was used at doses of 10 ng to 10 pg / kg tel. weight, parenterally, orally or topically as a thin layer of cream (i.e.: 1 pg / g neutral cream).

Given its good comparability with the situation in the human body, bepecin could be used to correct the disorders caused by the use of corticosteroids and / or even in some indications.

EXAMPLE 26. Hypertension, hypotension, and regulation of blood pressure

Experiments based on known and described methods, such as in: J. Physiol. Paris, 1993, 87 (5), 313-327 and Eur. J. Pharmacol., 1997,332 (1), 23-33, have shown that bepecin has no effect on blood pressure in normotensive animals, but may lower elevated blood pressure as well as raise too low blood pressure. Typically, bepecin lowers hypertension in Goldblatt hypertensive animals with two kidneys - 2K1C - or with one kidney - 1K1C - and the rat is fed diet high in fructose (80%) or on a high salt diet (15% ) over a long period; rat treated with NOS blocker LNAME (5 mg / kg, i.v.) or high dose KCI i.p. Bepecin also works against hypotension, that is, normalizes blood pressure in rats with chronic heart failure and elevated endothelin-1, with doxorubicin (2.5 mg / kg, ip, 6 times in 15 days) induced hypotension, in addition to reverse chronic cardiac cancellations. Therefore, bepecin works against hypotension in rats treated with the NO precursor L-arginine (100 mg / kg, i.v.). Bepecin was regularly given both prophylactically and therapeutic.

Bepecin, at doses of 10 ng to 10 pg / kg, parenterally or orally, causes a significant reduction or even disappearance of other lesions otherwise formed in these processes.

With hypovolumic shock, bepecin corrects blood pressure and prevents an otherwise fatal outcome. Thus, it is intended to increase blood volume loss, which can otherwise lead to death.

Given the significant similarity between the above models for blood pressure disorders and disorders in humans, the use of bepecin is justified in the treatment of blood pressure disorders, hypertension, hypotension and similar abnormalities.

EXAMPLE 27. Alcohol - Acute and Chronic Poisoning - Anesthetics

Studies on the efficacy of bepecin in alcohol poisoning have been made using the methods described in: Curr. Pharm. Des., 2010, 16 (10), 1224-1234; J. Physiol. Pharmacol., 2009, 60 (Suppl. 7), 177-181; Eur. J. Pharmacol., 1999, 364 (1), 23-31.

Bepecin at doses of 10 ng to 10 pg / kg tt, parenteral or oral, prevents and inhibits the effect of alcohol in the case of acute intoxication, addiction, acute and chronic gastric and liver wounds, as well as portal hypertension resulting from acute alcohol use, or due to prolonged chronic drinking of alcohol. By analogy, bepecin antagonizes the symptoms of addiction in morphine-dependent mice.

Additionally, beepecin, similar to naloxone (10 mg / kg, s.c.), antagonizes the anesthetic action of morphine (16 mg / kg, s.c.).

Taking alcohol as a prototype, bepecin counteracts the action of propofol, thiopental and ketamine. It reduces the effectiveness of both anesthesia and catalepsy (eg: fentanyl, dehydrobenzperidol).

Because alcohol consumption has similar effects in humans, bepecin can be used in both acute and chronic alcohol intoxication and withdrawal symptoms (including, for example, morphine and similar substances) and therefore anesthetics, where it regulates their anesthetic function.

EXAMPLE 28. Effect of bepecin on tumors

A common model for the study of experimental tumors is the tracking of the number of metastases of cancer and melanoma B-16 in mice. These experimental tumors bear great similarity to human tumors.

Ehrlich's ascitic tumor (EAT) can grow in all strains of mice and is widely used and recognized in these studies. Mice infected with EAT tumor cells survive up to a maximum of 25 days. Prior incubation of mice with bepecin (2 pg / ml) significantly prolonged the survival of the animals, mostly until the end of observation (45 days). Bepecin also prevents neutropenia, lowers reticulocytes, and improves hemoglobin levels after the administration of cyclophosphamide cytostatics.

In vitro experiments.

The effect on melanoma cells was investigated on human melanoma cell culture in RPMI-1840 medium with the addition of phosphate buffer and antibiotics. In the incubator, cell culture was formed at 37 ° C and in a humidified atmosphere with 5% CO ₂ Bepecin was added to well-developed cell culture at concentrations of 2 pg / ml 2 ng / ml and 10 ng / ml, especially in combination with vascular endothelial growth factor (VEGF - 10 ng / ml) for a period of 48 hours. Cell structure was observed in a light microscope and flow cytometry. Following the addition of bepecin to cell culture, the density of melanocytic cells decreased significantly. The cellular phenotype also changed from round melanocytic cells to spindle epithelial cells with fine cellular entanglement. The most prominent results were seen at a bepecin concentration of 10 ng / ml. Similar changes in the case of cells were first stimulated with VEGF and then with bepecin. Fig. 2.

Flow cytometry analysis showed that at a bepecin concentration of 2 pg / ml, the number of cells in the S phase was reduced to 20% relative to the control, at a bepecin concentration of 2 ng / ml and at 10 ng / ml to 55%.

Vestern imaging has shown that bepecin acts antimitogenically by inhibiting mitogen-activated protein kinase (MAPK), which responds to the VEGF factor.

Bepecin has a clear antitumor potential. Considering the similarities between animal models and human conditions and the favorable results obtained in vitro and in vivo, it will be successful in tumor therapy. It is also useful for reducing side effects in cytostatics

EXAMPLE 29. Wounds in the gastrointestinal tract

Methods and materials described in numerous publications: Curr. Pharm. Des., 2010, 16 (10), 1224-1234; J. Physiol. Pharmacol., 2009, 60 (Suppl. 7), 107-114; Dig. Dis. Sci., 2008, 108 (11), 7-17; Dig. Dis. Sci., 2009, 54 (1), 46-56. were used to determine the therapeutic effect of bepecin at doses of 10 pg to 10 ng / kg (ip, ig), by comparison with reference standards in several experimental ulcer models (for example: 24 hour restriction stress, subcutaneous or intrarectal cysteamine intake, 96 intake) percent ethanol to the stomach, NSAlAs lesions, DNFB (dinitrofluorobenzene) lesions, reflux esophagitis in pre-, concomitant and post-treatment Bepecin was administered either parenterally or orally.

On all models, bepecin effectively inhibited wound formation and promoted healing of existing wounds.

Given its strong resemblance to human disorders, bepecin could be successfully used in the treatment of all wounds in the gastrointestinal tract.

EXAMPLE 30. Potassium - hypokalemia and hyperkalemia

Potassium is crucial for the normal functioning of the smooth muscle of the heart, digestive tract, skeletal muscle and nervous system. Normal potassium concentration is very important for heart rhythm, as too low (hypokalemia) and too high (hyperkalemia) cause disturbances in cardiac rhythm and many other abnormalities in the body.

Using the methods described in J. Ciin. Exp. Cardiolog., 2012, 3 ^ 201 we followed the application of furosemide (100 mg / kg, ip) after electrocardiographically after 90-150 min electrocardiographically traced the duration of PR, RR, QRS and QT intervals, P, R, S, T waves and their amplitudes and beyond analyzed the occurrence of AV blockade, ventricular premature beats, and ventricular tachycardia. Despite the presence of hypokalemia, sinus rhythm was maintained in all animals treated with bepecin (10 pg / kg and 10 ng / kg, ip and ig), no premature ventricular beats, no ventricular tachycardia and AV blockade, no prolonged intervals and shorter waves. amplitude. Bepecin was given 90 min after furosemide (hypokalemia present, grade 3 AV blockade and / or ventricular tachycardia). Within 5-10 min, P, R, S T waves, duration of PR, RR, QRS, and QT intervals, RST amplitudes, total AV block were normalized in the bepecin group, and tachycardia was stopped.

According to the methods described in: J. Pharmacol. Sci., 2007, 104 (1), 7-18; J. Pharmacol. Sci., 2006, 102 (3), 269-277 demonstrated the full action of bepecin against KCI overdose (intraperitoneal (i), intragastric (II), and in vitro (III).

Therefore, bepecin would be useful as a basic therapy to normalize any abnormalities resulting from the presence of hypokalemia and hyperkalemia.

EXAMPLE 31 Calcium - Hypercalcemia

Calcium plays a very important role in the body because it acts on the cells of the heart muscle, blood vessels and neurons. The most common cause of high calcium levels in the body of hypercalcaemia is primary hyperparathyroidism, the second cause is malignancy and granulomatosis. Hypercalcaemia has many consequences such as hypertension, soft tissue and corneal calcification, and nephrolithiasis and peptic ulcer. Pathophysiological calcemia may be caused by diseases that increase osteoclast activity and bone reabsorption, which is not followed by the kidney. It can also occur with vitamin D overdoses and taking calcium supplements - antacids and thiazide diuretics.

A very common cause is the frequent use of calcium channel blockers in patients with hypertension.

Bepecin has been investigated for its function in hypercalcemia and in overdose of Ca channel blockers.

Calcium overdose and prolonged calcium deficiency cause acute pancreatitis and, in rats, shorten QT interval and prolong PQ. Rats received CaCb solution (200 mg and 400 mg / kg, i.p.). The ECG was continuously recorded and K, Na, Cl, amylase, creatine kinase and LDH were determined (at 5, 10, 15, 25 and 60 min.) And acute pancreatitis at 60 min as indicated in Dig. Dis. Sci., 1996, 41 (7), 1518-1526: Reg. Pept., 2009,

156 (1-3), 83-89; J. Pharmacol. Sci., 2004, 95 (1), 19-26. Bepecin was also given preventively (10 pg / kg, i.p.) 30 min before CaC ^.

Rats received bolus verapamil (40 mg / kg, i.p.). Bepecin (10 pg and 10ng / kg, i.p.) was administered 30 min before verapamil (pretreatment) or 5 min thereafter.

Bepecin works against the effects of severe calcemia, eliminates shortening of the QT interval and prolonging the PQ interval, works against otherwise severe acute pancreatitis, and reduces serum amylase.

Verapamil causes bradycardia (200 - 230 / min) after 5 minutes, together with a second stage of atrioventricular block lasting 30 and 45 min. Bepecin maintains and corrects sinus rhythm (atrioventricular block is also removed with therapeutic application).

Experiments have shown that bepecin prevents or reverses all the effects of hypercalcaemia. Therefore, it can be used to prevent this disorder.

EXAMPLE 32 Action on Myasthenic Syndrome

Myasthenic syndrome, which is reflected in increased muscle relaxation, was caused by a method known from Acta Neurol. Scand.Suppl., 1984, 100, 39-47 by intraperitoneal administration of magnesium sulfate overdose. Male and female rats, weighing 200-300 g, injected magnesium sulfate (500 mg / kg body weight) intraperitoneally to produce strong myasthenic syndrome (progressive weakness, powerlessness, muscle weakness), which progresses to 5 minutes complete immobility.

Bepecin administered 15 min before or at the same time as MgSO ₄ (10 pg / kg and 10 ng / kg body weight, ip). fully counteracts magnesium sulfate-induced disorders so that all animals remain normally active.

The pedestrian mill test shows that rats improve their skills after running bepecin and run longer than before

Thus, in general, bepecin could be the primary therapy for the normalization of disorders manifesting as myasthenic syndrome and with specific action against disorders occurring in magnesium therapy. Thus, bepecin could also be an essential therapy to improve the overall function of skeletal muscle.

EXAMPLE 33 Functioning in Neuromuscular and Neuropsychiatric Disorders

The dopamine system is widely considered to be important for the function of motor functions (J. Neural. Transm. 2010 Dec. 117 (12), 1359-69). Using the methods previously described in Life Sci. 2001 March 9, 68 (16), 1905-12 and J. Physiol.Parish, 2000 March-Apr., 94 (2), 105-110, we found that bepecin in a particular way strongly influences the dopamine system by works against the effects of dopamine receptor blockade, catalepsy, somatosensory disorders caused by neuroleptics such as haloperidol, flufenazine and clozapine, depletion of dopamine vesicles, or nigrostriatal destruction of dopamine. This is induced by the neurotoxin 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP). Bepecin greatly improves MPTP impaired somatosensory orientation and decreases MPTP-induced hyperactivity and, very importantly, MPTP motor anomalies (tremor, akinesia and catalepsy - otherwise very noticeable in the brine control group). It also eliminates the otherwise otherwise fatal outcome of the MPTP application in the control group.

As mentioned above, bepecin has a strong effect on the healing of muscle wounds after a cut or stroke, and on the healing of cut nerves, with or without anastomosis. Consequently, all of this implies that bepecin is capable of enhancing poor neuromuscular linkage, ie the transmission of signals from nerves to muscles.

It follows that bepecin could be used to improve impairment of muscle function in general.

Given the usual importance of these dopamine and serotonin models in animals and human disorders, bepecin could be used in the treatment of relevant neuropsychiatric abnormalities, especially related to the dopamine or serotonin system.

EXAMPLE 34 Insulin - Diabetes

Using the method described in Skin Pharmacol. Physiol., 2006, 19 (5), 266-274 and J. Physiol. Paris, 1999 Dec., 93 (6), 501-4, when administered bepecin topically results in a significant improvement in skin wound healing in diabetic animals. Bepecin has been found to inhibit the development of alloxan-induced ulcers, fructose-induced hypertension, and insulin resistance. In the same way, bepecin (10 pg / kg) injected (i) intraperitoneally or (ii) intragastrically directly behind insulin as an anti-ulcer peptide, in addition to gastric ulcers, has a strong effect on all disorders caused by insulin (in overdose: 250 lU / kg, ip) and fatal consequences (for example: seizures, possibly fatal, serious damage to neurons in the spinal cord and hippocampus, hepatomegaly, fatty liver, hepatic glycogen collapse with consequent severe hypoglycaemia).

Thus, the success of bepecin therapy clearly indicates the possible role of bepecin in controlling insulin, and that bepecin is likely to influence several consequences of insulin overuse. Therefore, the use of bepecin for the control of various disorders in diabetic patients could be possible, especially for abuse in insulin therapy.

EXAMPLE 35 Anti-inflammatory activity, pain and fever

Based on the articles published recently (J. Physiol. Paris, 1993, 87, 313-27, Inflammopharmacol., 2006, 14, 214-21; Curr. Pharm. Desd., Dec. 2010, 16, 1224-34, we demonstrated that bepecin decreases the release of inflammatory mediators (eg, myeloperoxidase, leukotriene B4, thromboxane B ₂ ) in vitro and in vivo. beepecin, as well as DNFB injury, as well as bepecin against temperature drop (immersion test) and temperature rise (yeast-induced) .Bepecin consistently works against both inflammatory pain (acetic acid) and anti-inflammatory pain (acetic acid). non-inflammatory pain (MgSO ₄ ).

Bepecin also raises the threshold for pain in the rat carrageenan test and shows an antihyperalgesic effect in the Randall-Selitte test.

Even with a sectioned sciatic nerve, without or with anastomosis, bepecin greatly reduces neuropathic pain.

The action of bepecin on chronically inflammatory lesions such as adjuvant arthritis and non-steroidal agents (NSAIDs) is very beneficial. In the treatment of well-developed adjuvant arthritis (caused by Freund's adjuvant), the effect of bepecin is clearly demonstrated after two weeks of treatment and can be clearly seen another year after administration.

Accordingly, bepecin could be a basic drug for reducing the negative effects of acute and chronic inflammation, for normalizing body temperature, and especially for reducing pain.

EXAMPLE 36 Functioning of Nerve Injuries

Using the method previously described in Regul. Pept., 2010 Feb.25, 160 (1-3), 3341, we showed healing of the sectioned sciatic nerve and improvement using bepecin (10 pg / kg, 10 ng / kg) after injury (intraperitoneal / intragastric / local at the site of anastomosis Improvement was seen clinically (autotomy), microscopic-morphometric and functional (EMG, one or two months after injury, improvement in gait at weekly intervals) Bepecin-treated rats showed faster axonal regeneration, which was determined histomorphometrically (increased density and size regenerated fibers, epineural and perineural regeneration, increased volume of myelinated fibers and myelin sheath, increased number of myelinated fibers per surface; electrophysiological as increased motor activity and functional (improved SFI factor).

Thus, bepecin significantly improves sciatic nerve healing in rats. Given the similarity between experimental models and human pathology, these findings would be practically useful in the treatment of various nerve injuries.

EXAMPLE 37 Somatosensory neurons, motor nerve damage, brain damage and interrupted nerve / muscle signal transduction

Both dysfunction and hyperfunction of somatosensory neurons are manifested in various abnormalities such as: congenital neurosensory neuropathy caused by diabetes, herpes zoster, postherpetic neuralgia, atopic dermatitis, impaired healing of damaged tissue, psoriasis, eczema, asthma, etc. Somatosensory neurons are the first line of defense system against damage, controlling homeostasis and triggering appropriate emergency response.

Their protective ability is evident in experimental skin and gastrointestinal mucosal damage with capsaicin. This substance degrades sensory fibers in high doses, while a low dose (below 0.5 mg / kg) activates neurotransmitter release and has a protective effect on the mucosa.

In the experiment described in Dig.Dis.Sci., 1996, 41 (8), 1604 -1614, a high dose of capsaicin was used to break down sensory fibers. Bepecin used at doses of 10 pg 10 ng / kg tel. animal weight, per os or locally, greatly reduces the effect of capsaicin.

Bepecin also greatly reduces the effects of brain injury in animals caused by falling weight. The model is described in: Regulat. Pept., 2010, 160 (1-3), 26 - 32.

Also, an experiment with the injection of succinylcholine (0.2 mg / kg) into the right muscle - quadriceps causes neuromuscular disruption of muscle function due to poor signal transmission from the nerve to the muscle. Bepecin the day before or after the injection of succinylcholine completely eliminates the local effect of succinylcholine.

Considering the neuroprotective action of bepecin, the similarity found between experimental models and human pathology, this could practically be an improvement in clinical practice for the treatment of various nerve injuries.

EXAMPLE 38 Action on encephalopathy and multiple sclerosis

Using the methods described previously (Eur. J. Pharmacol. 2011, 667, 322-9; Life Sci. 2011, 88, 535-42; J. Physiol. Pharmacol. 2010, 61, 241-50; J. Physiol. Pharmacol ., 2009, 60 Suppl 7, 107-14; Regul. Pept., 2010, 160,26-32): we found that bepecin works against all factors that subsequently lead to encephalopathy due to overdoses of NSAIDs. It also works against individual brain lesions resulting from insulin overdoses and against the toxic effect of life-threatening insulin (dangerous seizures, serious damage to neurons, spinal cord and hippocampus). Likewise, bepecin can reduce brain damage from traumatic wounds.

At present, natalizumab, a human monoclonal antibody with anti-cell adhesion activity of α4-integrin molecules, is used to treat multiple sclerosis and Crohn's disease.

Taking into account the efficacy of bepecin in various models of ulcerative colitis, the aforementioned efficacy of bepecin in the treatment of damaged muscles, nerves and brain, a possible effect of bepecin can also be expected in the relevant model for multiple sclerosis.

Among several different experimental models of allergic encephalitis, the cuprizone model was selected as the most reliable (J. Physiol. Pharmacol., 2013 and Brain, 2006, 129, 1940-1952). Vistar rats received 2.5% cuprizone in diet food combined with the addition of bepecin in drinking water (10 pg / kg or 10 ng / kg), 0.16 pg / ml / 12 ml / day / rat or 0.16 ng / ml / 12 ml / day / rat, to death after 4 days; additionally cuprizone 1 g / kg intragastric once daily and bepecin 10 pg / kg or 10 ng / kg intragastric. Thus, by using multiple, higher doses of cuprizone in food, we exaggerated and accelerated the formation of injuries. Damage occurred in different parts of the brain, most pronounced in the areas: corpus callosum, laterodorsal thalamus, and nucleus reunion. In rats treated with cuprizone and bepecin, there was consistently less nerve damage in all areas. The most favorable effect of bepecin was in those areas that were most affected.

It follows that, according to the experimental results, bepecin is suitable for the treatment of inflammatory bowel disease and multiple sclerosis. Also, the use of bepecin could be the primary therapy for various encephalopathies.

EXAMPLE 39 Antiviral activity

ARBO viruses, hepatitis, herpes and LCM viruses

Antiviral activity was tested in newborn mice of both sexes, 24 hours old, of BALB-C strain. Arboviruses (TBE = tick-borne encephalitis virus, Bhania, dengue types 1, 2, 3 and 4, Sindbis, West Nile and Chalovo), hepatitis A, (LCM) - lymphatic choriomeningitis virus and herpes type 1 and 2 viruses were used ic ( intracerebral) or after (oral) - in hepatitis A as a viral suspension at a dilution of 10 ' ² (0.02 ml / mouse). Doses were adjusted to be comparable to LD ₁₀ o at 0.02 ml ic (or pepper hepatitis A) / mouse and inoculum at a dilution of 10 ' ² . Bepecin (20 pg / kg body weight) or NaCI solution -0.9% (0.02 ml / mouse) were administered ic or ip as follows:

i - treatment 2 hours before viral infection (-2h), ii - simultaneously with viral infection (0), iii - 4 days later in the presence of symptoms of infection.

The results are shown in Table 7.

There were no signs of illness or death at the time of observation with bepecin administration prior to viral infection (s).

If bepecin was given at the same time as infection (ii), the outbreak occurred significantly later, after 20 days (in the control group without bepecin, all animals died after 5 days).

If bepecin was given in the presence of the first disease symptoms, the outbreak was significantly prolonged (iii).

The numbers in Table 7 represent the time when all infected animals die (including the control group). There were no fatalities in the second control group where only healthy animals were treated without treatment. The sign + indicates oral dosage, the sign a indicates arbovirus and the sign n.d. means there were no fatalities. All animals were observed 40 days after infection.

Tick-borne encephalitis virus

The efficacy of bepecin against tick-borne encephalitis virus in experimental mice is shown in Fig. Fig.3. Bepecin was administered at a dose of 0.02 mg / kg body weight. Bepecin significantly prolonged the survival of experimental animals and even prevented infection with adequate dosing.

To verify the virulence results of the viral suspension used, a portion of the brain tissue was removed with a viral suspension (TBE) and concurrently with the bepecin-treated animal and a brain homogenisate suspension was made. New animals were inoculated with this suspension and infected with a new viral suspension (TBE). The treated animals did not show any symptoms of the disease after 50 days, but the animals that received only brine instead of brain homogenate died within 5 days.

Table 7:

Kind of viruses: way input Time (days) to onset of death in mice after virus infection Animal treatment agent Brine 0.9% Bepecin Application 2 hours before viral infection <-2h) Application at the same time as a viral infection (0) Application 2 hours before viral infection (-2h) Application at the same time as a viral infection (0) 'P ic 'P ic 'P ic 'P ic TBE ³ 5 5 5 5 n.d. n.d. 20 20 Bhania ³ 5 5 5 5 n.d. n.d. 20 20 Dengue 1 ^a 5 5 5 5 n.d. n.d. 20 20 Dengue 2 ^a 5 5 5 5 n.d. n.d. 20 20 Dengue 3 ^a 5 5 5 5 n.d. n.d. 20 20 Dengue 4 ^a 5 5 5 5 n.d. n.d. 20 20 Sindbis ³ 5 5 5 5 n.d. n.d. 20 20 West Nile ^a 5 5 5 5 n.d. n.d. 20 20 Calovo ³ 5 5 5 5 n.d. n.d. 15 15 Hepatitis A + 5 5 5 5 n.d. n.d. 20 20 LCM ³ 5 5 5 5 n.d. n.d. 20 20 Herpes type HSV1 ^a 5 5 5 5 n.d. n.d. 20 20 Nothing (healthy animals) n.d n.d n.d n.d n.d. n.d. n.d n.d

Herpes virus

In a follow-up study using VERO cells (TICD ₅₀ , HSV-1 10 ¹⁰ , ID ₅ o / 2oopi and HSV2, 10 ¹¹ ID 5o / ioo μι, incubation for 24, 48 and 72 hours) we found that in-vitro The cellular system inoculated with HSV-1 and HSV-2 viruses, bepecin inhibits HSV reproduction and protects cells from the cytopathogenic action of HSV viruses.

The in vivo function of bepecin was determined by experiments on neonatal BALB / C mice with intracerebral inoculation with HSV-1 at 10 ¹ ° ID 50 / ιοομΐ and HSV-2 at 10 ¹¹ ID ₅₀ / ιοομι-Bepecin as a salt with arginine were administered at doses of 10 pg / kg and 100 pg / kg. Groups of untreated animals treated with saline and acyclovir at 10 mg / kg and 100 mg / kg were used as a comparison. All agents were administered 48 h and in the second group 72 h after viral infection. The results showed that bepecin in lethally infected mice significantly reduced the number of diseased animals and was more effective than the recognized acyclovir. The survival time in acyclovir-treated mice was 112/105/115 hours (mean, min, max.) And significantly longer for bepecin (at lower dose!): 131/128/135 hours.

We find that the efficacy of bepecin at doses of 10 pg / kg and 100 pg / kg is greater than that of acyclovir at significantly higher doses of 10 mg / kg and 100 mg / kg - under the same test conditions. Similar results were obtained in the case of CMV virus infection. Influenza virus A

A study of the action of bepecin on influenza A, type H1N1 and H3N2 viruses was performed on an MDCK model (Madin Darby Canine Kidney). The medium was MEM (Minimal Essential Medium) with the addition of 5% fetal bovine serum and antibiotics (Naruse N. et al., J. Antibiot., 1991, 44, 733-740). Bepecin exhibits a very potent inhibitory effect on influenza A viruses at a very low concentration of 32 pg / ml and is significantly more potent than previously known compounds used for this purpose.

Feline leukemia virus.

Feline leukemia virus (FeLV) is a retrovirus and as such is transmitted as an RNA virus but reversely transcribed into DNA. It is transmitted between cats with saliva or nasal secretions. The animal's immune system is no match for him, so the infection for the animal is fatal. A disease of this type is a form of blood lymphocyte cancer (leukemia).

The effects of bepecin (with the consent of the owners) have been studied in 32 cats (both sexes) from 6 months to 5 years of age who have not been previously vaccinated against the disease. They were all in a highly expressed stage of the disease, and the Combo FeLV / FIV test was positive. All animals had a high fever up to 41.5 ° C and had severe disease: icterus, diarrhea, tachycardia, abdominal pain, dehydration, and depression.

All cats received a subcutaneous injection of 1 pg / kg body weight (in 1 ml) immediately after detection of the disease, once daily for the first 8 days and then orally for 1 month. The improvement was immediately apparent, the temperature in the animals dropped by at least 1 ° C after 30 min, then gradually improved within 1 week. In some animals, the condition worsened again for a short period of 24 hours after 4 to 5 days, then improved again on the sixth day, followed by a full recovery in about 10 days.

Conclusion: Cats that have not been pre-vaccinated with cat feline leukemia can be fully cured within 10 days with a daily dose of bepecin 1 pg / kg body weight. So far, apart from vaccination and the very expensive interferon-omega, there has been no effective cure for this disease.

Based on the genetic similarity of the viral subtypes, we expect that bepecin would also be effective in the case of some other viral infections, such as varicella herpes virus (VZV or HHV-3), Epstein-Barr virus (EBV), human herpes viruses HHV-6, HHV- 7, HHV-8 (Kaposi) and Chikungunya (CHIKV).

Considering that these viruses cause similar disease events in humans, it is believed that bepecin can also be used to treat these diseases in humans, especially when the outlook for the patient is very unfavorable (eg AIDS and AIDS-related syndromes).

EXAMPLE 40. Acting on the sphincter

The experimental test method is described in detail in J. Pharmacol. Sci., 2007, 104 (1), 7-18 and J. Pharmacol. Sci., 2006, 102 (3), 269-277. We found that bepecin (10 pg / kg to 10 ng / kg m.p., parenterally or per os) in rats can maintain sphincter function (esophageal sphincter, pylorus sphincter and urethral sphincter) and pressure within the sphincter that would otherwise be disturbed. It can also quickly repair sphincter function and pressure, even with longer-lasting disorders (esophagitis, acute pancreatitis, transabdominal urolithiasis, and vaginal dilation). In addition, bepecin calms and eliminates esophagitis and other disorders that result from sphincter damage (acute pancreatitis).

Since there is a strong correlation of the model used with pathology in humans, bepecin could be used as a primary therapy for all disorders associated with sphincter malfunction.

EXAMPLE 41. Weight gain in animals - veterinary

Using the methods described in Dig. Dis. Sci., 2009, 54 (1), 45-56, studied rats with escalated short bowel syndrome and advanced weight loss after resection of the small intestine for 4 weeks. Postoperatively, the animals' exhaustion increased with a decrease in body weight, a twofold increase in crypt depth and a fourfold increase in muscle thickness in the first week, jejunal and ileal dilation, and impaired jejeunum / ileum ratio. Immediately after the administration of bepecin, a constant weight gain was recorded and also increased: fringe height, crypt depth, and muscle thickness (circular muscle layer). Rats treated with bepecin at doses of 10pg to 10 ng / kg bw, i.m. or p.o., had no difference in jejunum and ileum diameter. They had a constant jejunum / ileum ratio and an increased anastomosis break point.

Accordingly, young pigs treated with bepecin at doses of 1 to 21 days have a significantly better weight gain and are free of diarrhea. Bepecin could be used to achieve better weight gain and treatment of short bowel syndrome in farmed animals.

EXAMPLE 42. Effect of bepecin on sperm storage

It is well known that the process of freezing sperm during its storage results in an approximately 50% decrease in motility due to temperature change and osmotic influence. Morphological changes occur due to the organization, permeability and lipid composition of the sperm membrane.

We found a favorable effect of bepecin on the mobility of bull sperm, which was frozen by a slightly modified standard method (bepecin was added to the medium before freezing).

In the first part of the experiment, we determined the influence of the in-vitro dose of the most effective dose of bepecin, which is 20 ng / ml, if the sperm is so treated before freezing, given its quality after thawing. In the second part, the influence on the form of sperm. The results were evaluated by phase contrast microscope and computerized TIME processing.

Treatment with bepecin prior to freezing of the medium improves mobility after standing at

37 ° C relative to sperm control (p <0.05, subjective method: p = 0.0046, TIME: p = 0.014).

Concomitant treatment of sperm with bepecin immediately after melting improved motility relative to the control sample (p <0.05, subjective method: p = 0.0018, TIME: p = 0.014).

Based on these results, we conclude that the addition of bepecin to bull semen during freezing has a very good effect on the in vitro quality of sperm after thawing. An in-vivo study would be useful to confirm these results.

Conclusion: Bepecin already has a beneficial effect on the quality of sperm during freezing after its results.

EXAMPLE 43. Effect after removal of a blood vessel

Bepecin has a special protective effect after the removal of large blood vessels. Unlike the effect of L-NAME and L-arginine, bepecin quickly bridges the defective site between the blood vessels and thus reorganizes the blood supply quickly in the worst case scenario.

A portion of the femoral artery in rats (between a. Epigastrica caudalis and a. Femoralis circumflexa lateralis) was excised to investigate the presence of collateral blood vessels after critical vascular injury had occurred. The presence of collateral blood vessels within the defect was determined by the microangiography method described in Cardiovasc. Res., 1997 Sep., 35 (3), 547-52. An infrared camera was used for thermographic recording.

In the control group, no collateral blood vessels within the vascular defect were observed within 5 - 10 minutes after removal of a specific portion of the femoral artery in rats that received a local saline bath (1 ml / rat, 5 ml / kg) for 1 minute directly after vascular defect formation, Figure Fig. 5A.

Thin collateral veins are present but not interconnected, otherwise within the vascular defect 5-10 minutes after removal of a specific portion of the femoral artery in rats given a local bath of L-arginine solution (1 ml / rat, 100 mg / kg) over time 1 minute immediately after the onset of a vascular defect.

The area of vascular defect is completely empty, without collateral blood vessels within 5-10 minutes after removal of a certain part of femoral artery in rats receiving a local bath of L-NAME (1 ml / rat, 5 mg / kg) for 1 minute directly after the onset of a vascular defect.

In the case of bepecin, collateral blood vessels are present with complete interconnection in the form of a functional mesh that defects the defect (confirmed by thermographic imaging), 5-10 minutes after the removal of a particular part of the femoral artery in rats that have received a local bepecin bath (1 ml / rat, 10 pg / kg) for 1 minute immediately after the onset of vascular defect, Fig. 5B. This demonstrates that adaptation due to bepecin within existing collateral veins occurs rapidly after removal of the femoral artery and occlusion, with a view to restoring the passage of blood vessels and thus the flow of blood to the extremities.

Basically, later adaptation is an arteriogenesis, that is, the process of remodeling collateral blood vessels, while rapid adaptation is vasodilation (J. Vase. Surg. 2010 Jan. 51 (1), 165-73). However, the clear distinction between L-arginine and bepecin indicates that early adaptation (currently investigated during daily periods but not min periods) after vascular injury is not a simple vasodilation but a specific bridging process due to the use of bepecin. Thus, we can speak of an important process that greatly affects the initial increase in blood flow through collateral vessels so that it becomes permanent. This results in a very advanced stage and a significant improvement in end output (the formation of a vascular stem between the two stumps).

The results of the experiment show that bepecin could be the original therapy for disorders that require rapid reorganization of blood flow.

EXAMPLE 44 Acute toxicity

In all studies, bepecin has a very safe profile. Bepecin in the form of di-L-argininium salt (1: 2), as well as in the form of all other salts, administered to VVistar albino rats of both sexes, in very high doses, for example 1 g / kg body weight, intraperitoneally, intravenously or intragastric, shows no toxic and side effects.

Claims (12)

1. New stable salts of pentadecapeptide of general formula I: GlyGlu Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val. n.BAA 15 10 15 (SEQ D NO.1) wherein BAA is a basic amino acid and n is 1,2 or 3. 2. New pentadecapeptide stable salts of the general Formula I, wherein the basic amino acids are arginine, ornithine, lysine and others, preferably L-arginine. 3. A process for the preparation of a novel pentadecapeptide salt of the general formula I. characterized in that the pentadecapeptide of the sequence (SEQ ID NO. 1) is digested with the basic amino acids BAA: Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val + n BAA 15 10 15 SEQ ID NO 1 Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val. n BAA 15 10 15 SEQ ID NO 1 in a molar ratio of from 1 to 3 moles of basic amino acid per mole of pentadecapeptide (SEQ ID NO.1) in aqueous solution at room temperature, the solution is adjusted to the appropriate pH by titration, and then the resulting salt of formula I is isolated in solid form by HPLC chromatography and lyophilization. Process according to claim 3, characterized in that arginine, lysine, ornithine or another amino acid in the D, L or DL configuration, preferably L-arginine, is used as the basic amino acid. Process according to claims 3 and 4, characterized in that when the metabolism of 1 mol of basic amino acid with 1 mole of pentadecapeptide (SEQ ID NO. 1) is adjusted the pH of the solution from 4.1 to 5.0, preferably 4.60 + / -0.05. Process according to claims 3 and 4, characterized in that when the metabolism of 2 moles of basic amino acid with 1 mol of pentadecapeptide (SEQ ID NO. 1) is set the pH of the solution from 6.7 to 7.8, preferably 7.40 + /-0.05. Pharmaceutical formulation in solid and liquid form for oral, intravenous, rectal vaginal, intramuscular, topical and other uses containing a therapeutically effective amount of a pentadecapeptide salt of Formula I according to claims 1 and 2. Pharmaceutical formulation according to claim 7, characterized in that it contains, in addition to the pentadecapeptide salt of formula I, one or more other active substances, preferably antibiotics or antioxidants. Pharmaceutical formulation according to claims 7 and 8, characterized in that it contains stability enhancing additives from the group of alkali and alkaline earth carbonates or hydrogen carbonates, preferably sodium hydrogen carbonate. Pharmaceutical formulation according to claims 7, 8 and 9, characterized in that it contains stability enhancing additives selected from the group of sugars such as: D-mannitol, trehalose, sorbitol and the like, preferably D-mannitol. Pharmaceutical formulation according to claims 7 to 10 for use in the prevention, protection and treatment of the following diseases and conditions: - Stress related diseases such as gastrointestinal ulcers, inflammation, gactrointestinal tract inflammation, acute pancreatitis; - organoprotective action, protection and treatment of liver damage, protection of endothelium, prevention of adhesions; damage to the pancreas, prevention and treatment of myocardial infarction and stroke, protective function in the immune system; - viral infections, in particular hepatitis A viruses, herpes strains, influenza A strains and ARBO viruses such as: tick-borne encephalitis, West Nile, dengue types 1-4, cytomegalovirus CMV and LCM virus and feline leukenia virus; - for the treatment of melanoma and related tumors; - accelerated healing of wounds, burns, bone fractures, torn nerve connections, torn Achilles tendon, spinal cord injury, regeneration of muscle damage; - NO-related diseases and disorders: hypertension, hypotension, anaphylaxis, circulatory and septic shock, platelet aggregation; - treatment of neurological diseases and abnormalities: multiple sclerosis, myasthenia gravis, lupus erimatosis, dysfunction of somarosensory nerves, asthma, rhinitis, pemphicus and eczema; - catecholaminergic dysfunction, schizophrenia, dependence on amphetamine, alcohol and drugs; - prevention and elimination of problems caused by corticosteroids and NSAIDs; - treatment of wet macular degeneration; - as original therapy for disorders requiring rapid reorganization of blood circulation; - in veterinary medicine, to increase the weight gain of animals and to increase the stability of sperm during storage. Use of the pentadecapeptide salt according to claims 1 and 2 for the preparation of formulations serving as a dietary supplement.